The de novo formation of the spore wall during the process of sporulation in the budding yeast S. cerevisiae provides a powerful, tractable model for the study of fungal cell wall assembly. This grant is focused on elucidating the structure and mechanism of assembly of the outer two layers of the spore wall. These two layers, comprised of the polysaccharide chitosan and the polyphenol dityrosine, are absent from vegetative cells and enable spores to resist a variety of environmental stresses. Importantly, although chitosan and dityrosine are absent from vegetative cell walls in budding yeast they are present in the walls of many pathogenic fungi such as Candida albicans. Chitosan and dityrosine are not found in mammalian extracellular matrices, meaning that drugs may be found that specifically inhibit fungal cell wall assembly without affecting mammalian cells. The budding yeast spore wall provides an excellent model system to study fungal wall morphogenesis. Experiments in this grant are divided into three interrelated aims: (1) define the composition and structure of the outer spore wall and elucidate the biosynthetic pathways by which the components are synthesized and incorporated into the spore wall; (2) elaborate on our studies which have indicated that dolichol, an essential lipid required for glycosylation in vegetative cells, has a novel role in coordinating outer spore wall formation and (3) apply the knowledge gained from our studies of spore wall assembly to define the conserved genetic toolkit essential for construction of the chitosan/dityrosine module of in cell walls of Candida albicans.